Pertussis vaccines and methods of making and using

ABSTRACT

Bordetella pertussis  iron receptor proteins or portions thereof (e.g., one or more extracellular domains), alone or spliced into  B. pertussis  scaffold proteins (e.g., fimbrial or flagellin), are provided and can be used in acellular vaccines to protect against pertussis or other  Bordetella  diseases in humans and non-human mammals. In addition,  Bordetella  species grown under iron-starved conditions are provided and can be used in whole cell vaccines to protect against pertussis or other  Bordetella  diseases in humans and non-human mammals.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. 119(e) to U.S. Application No. 62/315,356 filed Mar. 30, 2016.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AI031088 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

This disclosure generally relates to vaccines.

BACKGROUND

Bordetella pertussis is the bacterial agent of whooping cough (pertussis). The primary pertussis vaccines currently in use in the U.S. are acellular (e.g., INFANRIX and BOOSTRIX (GlaxoSmithKline); DAPTACEL and ADACEL (Sanofi Pasteur) [infant and adult formulations for each, respectively]). The pertussis component of these vaccines contains detoxified pertussis toxin and two surface proteins (e.g., filamentous hemagglutinin, pertactin), while DAPTACEL and ADACEL also contain fimbrial (Fim) proteins, Fim2 and Fim3. For a number of different reasons (e.g., current acellular pertussis vaccines do not provide durable immunity; current acellular pertussis vaccines prevent disease symptoms but do not prevent B. pertussis colonization; and/or B. pertussis strains have been isolated from patients that lack a particular protein component of the vaccine), these acellular vaccines have been ineffective in preventing colonization and transmission of B. pertussis. Therefore, a new approach to impart immunity against B. pertussis is needed.

SUMMARY

Currently used acellular pertussis vaccines typically include a small set of antigens representing three or four virulence factors from B. pertussis. It is now apparent from the significant increase in pertussis cases and from epidemiological studies that these acellular vaccines lack the desired level of efficacy and duration of protection.

This disclosure provides a solution to this problem by describing polypeptides that can be used as vaccines to inoculate against one or more Bordetella species (e.g., B. pertussis, B. parapertussis, B. bronchiseptica, and/or B. avium). The vaccines described herein provide significantly improved immunity against B. pertussis over existing acellular pertussis vaccines. Further, a similar strategy can be applied to provide a whole-cell pertussis vaccine that imparts improved immunity relative to the whole-cell pertussis vaccines that are used in many countries throughout the world.

In one aspect, a chimeric polypeptide is provided that includes at least one antigenic polypeptide and a scaffold protein.

In some embodiments, the at least one antigenic polypeptide includes an iron receptor protein or an antigenic portion thereof. In some embodiments, the antigenic portion of an iron receptor protein includes at least one extracellular domain. In some embodiments, the iron receptor protein is a TonB-dependent receptor protein or an antigenic portion thereof. A representative TonB-dependent receptor protein is a ferric enterobactin siderophore (BfeA) receptor protein. In some embodiments, the iron receptor protein is a hemin or hemoprotein receptor or an antigenic portion thereof. A representative hemin or hemoprotein receptor is a BhuR protein. In some embodiments, the iron receptor protein is a siderophore receptor or an antigenic portion thereof. A representative siderophore receptor is an alcaligin siderophore receptor (FauA).

In some embodiments, the scaffold protein is a fimbrial protein or a flagellin protein. Representative fimbrial proteins include, without limitation, a fimbrial 2 protein or fimbrial 3 protein. A representative flagellin protein is a flagellin subunit protein.

In one aspect, a nucleic acid molecule encoding a polypeptide as described herein (e.g., a chimeric polypeptide) is provided. In another aspect, a construct that includes such a nucleic acid molecule is provided. In still another aspect, a host cell that includes such a nucleic acid molecule or construct is provided.

In another aspect, an acellular B. pertussis vaccine for protecting a subject against infection by B. pertussis, B. parapertussis, B. bronchiseptica and/or B. avium is provided. Such a vaccine typically includes a chimeric polypeptide as described herein and a pharmaceutically acceptable carrier. In some embodiments, such an acellular vaccine further includes an adjuvant.

In still another aspect, a whole cell B. pertussis vaccine for protecting a subject against infection by B. pertussis, B. parapertussis, B. bronchiseptica and/or B. avium is provided. Such a vaccine typically includes a composition of B. pertussis grown under iron-starvation conditions and a pharmaceutically acceptable carrier. In some embodiments, such a whole cell vaccine further includes an adjuvant.

In yet another aspect, a method of vaccinating a subject against B. pertussis is provided. Such a method typically includes administering a polypeptide as described herein (e.g., a chimeric polypeptide) or a vaccine as described herein to a subject. Representative subjects include, without limitation, humans, canines, pigs, rabbits, cats, or birds.

In still another aspect, a method of making an acellular B. pertussis vaccine is provided. Such a method typically includes providing a polypeptide as described herein (e.g., a chimeric polypeptide); or expressing a nucleic acid molecule or construct as described herein; or culturing a host cell as described herein (e.g., a host cell expressing a nucleic acid molecule or construct as described herein); and combining the polypeptide produced therefrom with a pharmaceutically acceptable carrier. In some embodiments, such a method further includes adding an adjuvant.

In yet another aspect, a method of making a whole-cell B. pertussis vaccine is provided. Such a method typically includes culturing B. pertussis under iron-starvation conditions; and processing the B. pertussis grown under iron-starvation conditions into a whole-cell vaccine. In some embodiments, such a method further includes adding an adjuvant.

In one aspect, a polypeptide that includes at least one extracellular domain of an iron receptor protein is provided. In some embodiments, the iron receptor protein is a heme, hemin or hemoprotein receptor (e.g., a BhuR protein). In some embodiments, the iron receptor protein is a siderophore receptor (e.g., an alcaligin siderophore receptor [FauA]). In some embodiments, the iron receptor protein is a TonB-dependent receptor protein (e.g., an enterobactin siderophore receptor protein [BfeA]).

In another aspect, a chimeric polypeptide that includes a scaffold protein or portion thereof and at least one antigenic polypeptide is provided. In some embodiments, the at least one antigenic polypeptide is spliced into a scaffold protein. Representative scaffold proteins include a fimbrial 2 or a fimbrial 3 protein or a flagellin protein. In some embodiments, the at least one antigenic polypeptide includes at least one extracellular domain of an iron receptor protein. In some embodiments, the at least one antigenic polypeptide includes an iron receptor protein. In some embodiments, the at least one antigenic polypeptide includes at least one extracellular domain of a TonB-dependent receptor protein. In some embodiments, the at least one antigenic polypeptide includes a TonB-dependent receptor protein.

In still another aspect, a nucleic acid molecule is provided that encodes any of the polypeptides described herein. In yet another aspect, a construct is provided that includes a nucleic acid molecule as described herein. In another aspect, a host cell is provided that includes a nucleic acid molecule as described herein or a construct as described herein.

In one aspect, an acellular B. pertussis vaccine is provided for protecting a subject against infection by B. pertussis, B. parapertussis, B. bronchiseptica and/or B. avium. Such a vaccine typically includes any of the polypeptides described herein. In another aspect, a whole cell B. pertussis vaccine is provided for protecting a subject against infection by B. pertussis, B. parapertussis, B. bronchiseptica and/or B. avium. Such a vaccine typically includes B. pertussis grown under iron-starved conditions that promote the production of iron receptors. In some embodiments, a vaccine further can include an adjuvant.

In still another aspect, a method of vaccinating a subject against B. pertussis is provided. Such a method typically includes administering, to a subject, any of the polypeptides described herein, any of the nucleic acid molecules described herein, any of the constructs described herein, any of the host cells described herein, or any of the vaccines described herein. In some embodiments, the subject is a human, a canine, a pig, a rabbit, a cat, or a bird.

In still another aspect, a method of making an acellular B. pertussis vaccine is provided. Such a method typically includes providing any of the polypeptides described herein; or expressing any of the nucleic acid molecules described herein; or expressing any of the constructs described herein; or culturing any of the host cells described herein; and combining the polypeptide produced therefrom with a pharmaceutically acceptable carrier. In yet another aspect, a method of making a whole-cell B. pertussis vaccine is provided. Such a method typically includes culturing B. pertussis under iron-starvation conditions that enhance iron receptor production; and processing the iron-starved B. pertussis cells for delivery as a vaccine. In some embodiments, such a method further includes adding an adjuvant.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

DESCRIPTION OF DRAWINGS

FIG. 1A is a photograph of an immunoblot showing total membranes proteins prepared from B. pertussis (Bp) and B. bronchiseptica (Bb) cultured under iron-replete (+Fe) and iron-depleted (−Fe) conditions probed using human sera from normal uninfected donors and from infected pertussis patients.

FIG. 1B is a photograph showing the juxtaposition of B. pertussis FIG. 1A pertussis patient immunoblot lanes; immunoreactive iron-regulated proteins are indicated with small triangles.

FIG. 1C is a photograph showing total membrane proteins of B. pertussis and B. bronchiseptica, and outer membrane proteins from B. bronchiseptica strains that overproduce the FauA, BhuR or BfeA receptors of B. pertussis, probed using sera from normal and culture-positive donors (different donors than in FIG. 1A).

FIG. 2 is a graph showing that B. pertussis mutants lacking the FauA and BhuR receptors are attenuated in competition infections using a mouse model of pertussis. Mice (5/group) were co-infected with a 1:1 mixture of wild type B. pertussis and a receptor mutant derivative. Bacterial colony forming units (CFU) of each strain pair in the lungs and trachea were determined at different times post-infection. The competitive index is the ratio of the mutant/wild-type CFU ratio recovered at each time point divided by the mutant/wild-type CFU ratio in the input inoculum (mean values shown). A competitive index of <1 indicates the mutant strain is less fit (exhibits less growth in mice) compared with the wild type parent strain. A plot (grey line) showing CFU levels typical of B. pertussis mouse respiratory tract infections is overlaid for reference. A B. pertussis fauA mutant is defective for growth in the mouse throughout infection and a bhuR mutant is most defective during late infection. A double mutant lacking both fauA and bhuR is avirulent.

FIG. 3 is a schematic showing the structural domains of the B. pertussis FauA and BhuR receptor proteins, including the cell surface-exposed loops that connect the antiparallel transmembrane beta strands (wide yellow ribbons). Both proteins have 11 flexible extracellular loops (within dashed boxes in structures, and dark regions within “Barrel” region). BhuR has an additional N-terminal extension (NTE) involved in cell surface signaling. A linear depiction of the loop domain locations is shown below the structures.

FIG. 4A shows a photograph of a SDS-PAGE gel showing purification of polyHis-tagged Bp FauA protein produced in Escherichia coli. M, molecular mass markers; Total, total cell proteins; 1-4, eluate fractions from immobilized metal affinity chromatography.

FIG. 4B shows a photograph of an immunoblot using rabbit antiserum raised to recombinant FauA to probe outer membrane proteins of iron-starved B. pertussis strains. WT, wild-type B. pertussis; ΔfauA, B. pertussis fauA in-frame deletion mutant; ΔfauA/fauA+, B. pertussis ΔfauA mutant complemented using the wild type fauA gene carried on a plasmid.

FIG. 5A is a schematic showing an expression system for production of Fim3 and Fim3-iron receptor chimeras in B. pertussis.

FIG. 5B is a schematic showing gene assembly using a thermostable ligase cycling reaction.

FIG. 6 is a schematic showing a map of B. pertussis Fim3 functional domains. Regions predicted to be permissive for foreign epitope insertion are shown in red.

FIG. 7A is a photograph of a SDS-PAGE gel showing Fim3 and two Fim3-BhuR loop domain chimeric proteins produced in E. coli. M, molecular mass markers; Vec, vector control; Fim3, Fim3 of B. pertussis; L3, Fim3 with BhuR loop 3; L4, Fim3 with BhuR loop 4; BhuR, outer membrane proteins of BhuR-over-producing B. pertussis cells.

FIG. 7B is a photograph of an immunoblot of a duplicate gel (as shown in FIG. 7A) probed with BhuR-specific rabbit antiserum, demonstrating reactivity with the Fim3-BhuR loop 3 and Fim3-BhuR loop 4 chimeric proteins.

FIG. 8A is a schematic showing a model of multivalent Fim3-iron receptor chimeric protein filaments on the B. pertussis cell surface. Grey ovals, Fim3 fimbrial subunits; red and yellow spheres, iron receptor loop domains displayed on the fimbrial scaffold.

FIG. 8B is a photograph of a SDS-PAGE gel showing extraction and purification of overproduced B. pertussis Fim3 protein filaments. M, molecular mass markers; Extract, urea extract of B. pertussis cells; PEG, Fim3 precipitated from urea extract using PEG 600; Sol, Fim3 filaments after solubilization of PEG 600 precipitate (Fim3, ca. 20 kDal).

FIG. 9A is a map of a plasmid for production of flagellin-iron receptor chimeric proteins in E. coli, with inducible expression driven from the tac promoter under LacI repressor control. The site for in-frame insertion of iron receptor loop encoding DNA segments within fliC is shown.

FIG. 9B is a model of a flagellin-iron receptor chimeric protein monomer, showing the dispensible region of FliC that is permissive to peptide insertions or substitutions, and the polymerization domains involved in self-assembly of the flagellar filament. The loop peptide is shown in red.

FIG. 9C shows a top view of proposed hybrid flagellar filaments having 11 subunits per turn and with exposed iron receptor loops (filaments displaying a single or multiple different iron receptor loops are shown).

FIG. 9D is a side view of proposed hybrid flagellar filaments shown in FIG. 9C.

DETAILED DESCRIPTION

The mammalian host is a very iron-poor environment; in order to multiply and cause disease, bacterial pathogens must use their iron uptake systems to overcome host iron restriction. Such iron uptake systems include bacterial cell surface proteins (e.g., receptors) that help take up iron-containing heme from the host or iron-loaded bacterial siderophores. Such proteins, however, have not been previously used as pertussis vaccine antigens. The immune response to vaccination using these receptor proteins (or antigenic domains thereof) would target essential bacterial processes by blocking iron uptake, thus preventing the establishment of infection, and also by promoting clearance of the organism. By targeting iron uptake systems that Bordetella or other genera of bacteria requires at both early and later stages of infection, more effective bacterial clearance and protection can be achieved.

This strategy can be used to produce acellular pertussis vaccines. Acellular vaccines typically are preferred in the U.S. Therefore, polypeptides are provided herein that include at least one domain (e.g., an extracellular domain) from a protein that is induced in response to low iron availability or iron starvation. These proteins typically are receptors that transport iron-bound molecules (e.g., siderophores or heme) or iron-carrying proteins, or just the iron, into the cell. These proteins are referred to herein as “iron receptor proteins.” Such iron receptor proteins, or portions thereof, can be used as antigens in vaccine compositions.

There are a number of different iron receptor proteins that can be used in an acellular pertussis vaccine as described herein. Simply by way of example, an iron receptor protein can be a receptor for a heme protein, or a receptor for a siderophore, or a receptor for another iron source or nutrient that is also a TonB-dependent receptor. A heme receptor includes, without limitation, a BhuR protein (e.g., gene locus tag BP0347); a siderophore receptor includes, without limitation, an alcaligin siderophore receptor (FauA; e.g., gene locus tag BP2463) or an enterobactin siderophore receptor (BfeA; e.g., gene locus tag BP2901), or a catechol or catecholamine receptor (e.g., BfrA, BfrD, BfrE); and a TonB-dependent receptor includes, without limitation, TonB-dependent receptors for iron as well as TonB-dependent receptors for one or more nutrients or co-factor (see, for example, Table 1).

TABLE 1 Bordetella TonB-dependent outer membrane receptors Characterized receptors of known substrate specificity Receptor name Locus Tag^(a) Iron Source Substrate FauA BP2463, BPP3450, BB3900 alcaligin BfeA BP2901, BPP2495, BB1942 enterobactin BhuR BP0347, BPP4185, BB4655 heme BfrA BB4761 catecholamine, catechol BfrD BP0856, BPP3376, BB3826 catecholamine, catechol BfrE BP0857, BPP3375, BB3825 catecholamine, catechol Predicted receptors of unknown substrate specificity Receptor name Locus Tag^(a) BfrB BP2016, BPP2396, BB1846 BfrC BP3663, BPP0079, BB0078 BfrF BP0736, BPP3688, BB4122 BfrG BP2922, BPP1078, BB1294 BfrH BP1138, BPP3206, BB3658 BfrI BP1962, BPP2334, BB1785 BfrZ BB4744 HemC BP0456, BPP4370, BB4956 — BPP2457, BB1905 — BP3101, BPP0746, BB0832 — BP3595, BPP3984, BB4457 — BP3077, BPP0186, BB0189 — BP1760, BPP1991, BB2179 ^(a)BP, B. pertussis; BPP, B. parapertussis; BB, B. bronchiseptica

TonB-dependent receptors are bacterial outer membrane proteins that bind and transport ferric iron chelates (siderophores), heme, vitamin B₁₂, as well as other substrates. High affinity transport of TonB-dependent receptor substrates across the outer membrane requires a proton-motive force, with the energy transduced by the TonB-ExbB-ExbD inner membrane protein complex. The structures of twelve TonB-dependent receptors have been solved (some alone, some with bound ligands, and some complexed with TonB), and forty-five crystal structures now exist for comparison. All known TonB-dependent receptors have the same domain architecture, and it is likely that all TonB-dependent receptors have this general structure—they all have a twenty-two-stranded transmembrane beta-barrel surrounding a globular plug domain, and a TonB-interacting domain. The twenty-two beta-strands that traverse back and forth across the outer membrane to form the beta-barrel are joined on the extracellular and periplasmic aspects by loop segments. The extracellular loop segments, along with residues on the extracellular side of the plug domain, form the specific ligand binding sites. These extracellular loops are surface-exposed, solvent-accessible, and highly flexible, and are specific for binding their cognate substrate. The strong conservation of TonB-dependent receptor domain architecture allows for accurate prediction of the transmembrane beta-strands and the extracellular loop domains using freely available protein modeling algorithms such as PRED-TMBB, I-TASSER, ROBETTA, and SPARKS-X starting with known TonB-dependent receptor structural data as templates.

Experimental results (see, for example, FIG. 2) show that FauA is needed for B. pertussis growth throughout the course of mouse infection, and BhuR is required for growth during late infection, when host heme sources become available due to tissue damage. Thus, elimination of both FauA and BhuR renders B. pertussis avirulent Targeting by vaccines of nutrient receptors that are important during different stages of infection provides broader protection against the bacteria not only attempting to colonize, but also against those already colonizing and persisting in the respiratory tract.

One or more of the iron receptor proteins, or portions thereof, described herein for use as antigens in vaccine compositions can be displayed within one or more scaffold proteins. Scaffold proteins provide the benefit of increased antigen multivalency, which imparts enhanced immunogenity. Multivalent antigens more effectively crosslink adjacent B-cell receptors (antigen-specific surface immunoglobulins) to initiate the signaling cascade that induces B-cell proliferation and differentiation. Furthermore, the extended crosslinking confers high avidity on the antigen-B-cell interaction. Multivalent antigens displaying a particular receptor loop domain on a polymeric fimbrial or flagellar scaffold can be used singly as vaccine antigens, or they can be dissociated into monomeric subunits, mixed, and reassorted to produced multivalent antigens displaying multiple different receptor loop domains. These polyvalent antigens can be used to generate immune responses to multiple distinct receptor loop domains.

A scaffold protein can be one or more fimbrial (Fim) or flagellin proteins into which specific regions or domains of an antigenic polypeptide are introduced. B. pertussis Fim2 and Fim3 proteins are already currently used in some existing acellular vaccine formulations. For example, an antigenic polypeptide or portions thereof (e.g., a first antigenic polypeptide or portions thereof, a second antigenic polypeptide or portions thereof, optionally, a third antigenic polypeptide or portions thereof, a fourth antigenic polypeptide or portions thereof, etc., etc.) can be spliced into regions of the fimbrial protein such that the fimbrial protein acts as a scaffold to display the antigenic polypeptide(s). Representative fimbrial proteins include, without limitation, fimbrial protein X (“FimX”), fimbrial protein 2 (“Fim2”) or fimbrial protein 3 (“Fim3”). Without limitation, the sequences of various fim genes and Fim proteins can be found, e.g., in GenBank Accession Nos. BP1568, BP1119, BP2674, and their orthologs in other Bordetella species.

Flagellin proteins are the major subunits of bacterial flagella, and have been used to produce vaccine antigens for many viral and bacterial agents. Flagellin proteins have broadly conserved structural features, and large insertions that replace the variable solvent-exposed region of E. coli flagellin are remarkably well tolerated without affecting flagellar export, assembly, or function. In addition to providing high antigen valence, flagellin polymers are recognized by the innate immune system as a pathogen-associated molecular pattern molecule, stimulating adaptive immunity. Representative flagellin proteins include, e.g., FliC and FlaA, and, without limitation, the sequences of various flagellin genes and flagellin proteins can be found, e.g., in GenBank Accession Nos. BP0996 and its orthologs in other Bordetella species, and b1923 and its orthologs in E. coli and Salmonella spp.

One way to accomplish this is to express one or more antigenic proteins, or portions thereof, and a scaffold protein in one or more chimeric polypeptides. Such chimeric polypeptides can be genetically constructed using recombinant methods known in the art. Such chimeric polypeptides result in polymeric, multivalent antigens that are highly effective vaccines. As discussed herein, an antigenic polypeptide or a portion thereof can be one or more of the iron receptor proteins described herein or a portion thereof (e.g., one or more of the TonB-dependent receptor proteins described herein, or a portion thereof).

Polypeptides are provided herein that can be used as vaccines against B. pertussis or other Bordetella species. As indicated herein, a polypeptide used in a vaccine can be an iron receptor protein or a chimeric polypeptide that includes at least one scaffold protein into which an antigenic polypeptide has been inserted (e.g., an iron receptor protein and/or another antigenic polypeptide). As used herein, a “purified” polypeptide is a polypeptide that has been separated or purified from cellular components that naturally accompany it. Typically, the polypeptide is considered “purified” when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dry weight, free from the polypeptides and naturally occurring molecules with which it is naturally associated. Since a polypeptide that is chemically synthesized is, by nature, separated from the components that naturally accompany it, a synthetic polypeptide is “purified.”

In addition, nucleic acids encoding such polypeptides are provided herein. As used herein, nucleic acids can include DNA and RNA, and includes nucleic acids that contain one or more nucleotide analogs or backbone modifications. A nucleic acid can be single stranded or double stranded, which usually depends upon its intended use. As used herein, an “isolated” nucleic acid molecule is a nucleic acid molecule that is free of sequences that naturally flank one or both ends of the nucleic acid in the genome of the organism from which the isolated nucleic acid molecule is derived (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease digestion). Such an isolated nucleic acid molecule is generally introduced into a vector (e.g., a cloning vector, or an expression vector) for convenience of manipulation or to generate a fusion nucleic acid molecule, discussed in more detail below. In addition, an isolated nucleic acid molecule can include an engineered nucleic acid molecule such as a recombinant or a synthetic nucleic acid molecule.

Polypeptides can be purified from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. A polypeptide also can be purified, for example, by expressing a nucleic acid in an expression vector. In addition, a purified polypeptide can be obtained by chemical synthesis. The extent of purity of a polypeptide can be measured using any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Similarly, nucleic acids can be isolated using techniques routine in the art. For example, nucleic acids can be isolated using any method including, without limitation, recombinant nucleic acid technology, and/or the polymerase chain reaction (PCR). General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides.

Also provided are nucleic acids and polypeptides that differ in sequence from the wild type sequence. Nucleic acids and polypeptides that differ in sequence from the corresponding wild type sequence can have at least 50% sequence identity (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the corresponding wild type sequence. In calculating percent sequence identity, two sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence. It also will be appreciated that a single sequence can align with more than one other sequence and hence, can have different percent sequence identity values over each aligned region.

The alignment of two or more sequences to determine percent sequence identity can be performed using the computer program ClustalW and default parameters, which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). See, e.g., Chenna et al., 2003, Nucleic Acids Res., 31(13):3497-500. ClustalW calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the default parameters can be used (i.e., word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5); for an alignment of multiple nucleic acid sequences, the following parameters can be used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of polypeptide sequences, the following parameters can be used: word size: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; and gap penalty: 3. For multiple alignment of polypeptide sequences, the following parameters can be used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific gap penalties: on. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher website or at the European Bioinformatics Institute website on the World Wide Web.

A construct, also referred to as a vector, containing a nucleic acid (e.g., a nucleic acid that encodes a polypeptide as described herein for use in a vaccine) is provided. Constructs, including expression constructs, are commercially available or can be produced by recombinant DNA techniques routine in the art. A construct containing a nucleic acid can have expression elements, and further can include sequences such as those encoding a selectable marker (e.g., an antibiotic resistance gene). A construct containing a nucleic acid can be fused to a second polypeptide that, for example, can be used in purification of the encoded polypeptide (e.g., a 6× tag polypeptide, a glutathione S-transferase (GST) polypeptide)

Expression elements include nucleic acid sequences that direct and regulate expression of nucleic acid coding sequences. One example of an expression element is a promoter sequence. Expression elements also can include introns, enhancer sequences, response elements, or inducible elements that modulate expression of a nucleic acid. Expression elements can be of bacterial, yeast, insect, mammalian, or viral origin, and vectors can contain a combination of elements from different origins. As used herein, operably linked means that a promoter or other expression element(s) are positioned in a vector relative to a nucleic acid in such a way as to direct or regulate expression of the nucleic acid. Expression elements in a construct can be operably linked to a coding sequence in cis or in trans; expression elements that are operably linked to a coding sequence in trans may be in-frame with the coding sequence.

Constructs as described herein can be introduced into a host cell. As used herein, “host cell” refers to the particular cell into which a construct is introduced and also includes the progeny of such a cell that carry the construct. A host cell can be any prokaryotic or eukaryotic cell. For example, nucleic acids can be expressed in bacterial cells such as E. coli, or in insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. Many methods for introducing nucleic acids into host cells, both in vivo and in vitro, are well known to those skilled in the art and include, without limitation, electroporation, calcium phosphate precipitation, polyethylene glycol (PEG) transformation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer.

A similar strategy that takes advantage of the pertussis bacterial requirement for iron can be used to produce whole-cell pertussis (wP) vaccines. Whole-cell vaccines typically are preferred in many non-U.S. countries around the world. Similar to the strategy described above for acellular vaccines, B. pertussis grown under iron-starvation conditions can be used in a whole-cell vaccine. Since the whole-cell pertussis vaccines currently being used are produced by growing bacterial cells in iron-rich medium, the bacteria in those vaccines will not have produced their iron receptors and will lack those important antigens. On the other hand, iron-starved B. pertussis produces abundant amounts of iron uptake receptors. For example, alcaligin siderophore production is evidence of iron starvation in B. pertussis. Alcaligin siderophore production level can be assessed using a siderophore detection assay such as a chrome azurol S universal siderophore assay, or using growth stimulation assays of siderophore-deficient mutant Bordetella indicator strains. Iron receptor protein production levels in iron-starved B. pertussis cells can be assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of bacterial cell proteins, and/or by immunoblot analysis using iron receptor specific antisera. As with the acellular vaccines, an immune response against the iron uptake receptors targets essential bacterial processes by blocking iron uptake, preventing a productive infection and promoting immune clearance.

Any of the polypeptides described herein (e.g., an iron receptor protein or a chimeric polypeptide including a scaffold protein and an antigenic polypeptide) can be used in an acellular vaccine to protect a subject against infection by B. pertussis. Similarly, the iron-starved B. pertussis described herein can be used in a whole-cell vaccine to protect a subject against infection by B. pertussis.

Vaccines are well-known in the art, and often include a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” is intended to include any and all excipients, solvents, dispersion media, coatings, antibacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like, compatible with administration. Pharmaceutically acceptable carriers for delivering compounds are well known in the art. See, for example Remington: The Science and Practice of Pharmacy, University of the Sciences in Philadelphia, Ed., 21^(st) Edition, 2005, Lippincott Williams & Wilkins; and The Pharmacological Basis of Therapeutics, Goodman and Gilman, Eds., 12^(th) Ed., 2001, McGraw-Hill Co. The type of pharmaceutically acceptable carrier used in a particular formulation can depend on various factors, such as, for example, the physical and chemical properties of the compound, the route of administration, and the manufacturing procedure. Vaccines often include an adjuvant, in addition to the primary antigen, to further increase the immune response by the subject.

The vaccines described herein (e.g., an acellular vaccine including an iron receptor protein or a chimeric polypeptide including a scaffold protein and an antigenic polypeptide or a whole-cell vaccine including iron-starved B. pertussis cells) can be used in a method of vaccinating or immunizing a subject against a B. pertussis infection (e.g., a productive infection). Additionally or alternatively, vaccinating or immunizing a subject can prophylactically protect the subject against infection by B. pertussis.

The vaccines described herein can be administered in an effective amount to a subject. Typically, an effective amount is an amount that prevents or treats a Bordetella infection in a subject without inducing any adverse effects. The amount of polypeptide in each dose of vaccine typically is the minimal amount that induces an immunoprotective response in a subject without significant, adverse side effects. The particular amount of polypeptide in a vaccine will vary depending upon the antigenicity of the polypeptide as well as the presence of any adjuvant. In some instances, a vaccine dose includes between about 1 μg and about 1000 μg of protein (e.g., about 1 μg to about 200 μg; about 1 μg to about 50 μg; or about 1 μg to about 10 μg of protein) in a volume of about 50 μl to about 2 ml per dose (e.g., about 100 μl to about 1.5 ml; about 250 μl to about 1 ml; about 100 μl to about 500 μl; or about 100 μl to about 250 μl). A whole cell vaccine dose is standardized to Opacity Units using a WHO reference preparation (IU), with one vaccine dose not exceeding 20 IU.

The vaccines described herein can be administered to any subject that can be infected with a Bordetella species. For example, B. pertussis and B. parapertussis infect humans, while B. bronchiseptica occasionally infects humans but also infects other mammals such as canines and felines (causing kennel cough) and pigs (causing atrophic rhinitis) and B. avium infects birds (causing turkey rhinotracheitis). Alternatively, the strategy described herein can be used to make an acellular or whole-cell vaccine from another Bordetella species. A vaccine as described herein typically is formulated to be compatible with its intended route of administration. Suitable routes of administration include, without limitation, intranasal, oral, topical, pulmonary, ocular, intestinal, and parenteral administration. Routes for parenteral administration include intravenous, intramuscular, and subcutaneous administration, as well as intraperitoneal, intra-arterial, intra-articular, intracardiac, intracisternal, intradermal, intralesional, intraocular, intrapleural, intrathecal, intrauterine, and intraventricular administration. For example, a vaccine as described herein can be delivered subcutaneously.

The vaccines described herein can be made using methods that are known in the art. For example, the acellular vaccines described herein can be made by combining any of the polypeptides described herein (e.g., an iron receptor protein or a chimeric polypeptide including a scaffold protein and an antigenic polypeptide) with a suitable pharmaceutically acceptable carrier. A polypeptide for use in a pertussis vaccine as described herein can be purified, or a polypeptide for use in a pertussis vaccine as described herein can be expressed from an appropriate nucleic acid (e.g., contained within a construct; contained within a host cell).

In addition, the whole-cellular vaccines described herein can be made by combining iron-starved B. pertussis cells (e.g., B. pertussis cells cultured under reduced iron or iron-starvation conditions) with a pharmaceutically acceptable carrier. Iron starvation can be achieved by sub-culturing B. pertussis from iron-replete culture media to culture media lacking iron supplementation, or by the addition of non-utilizable iron chelators to iron-replete culture medium. As discussed herein, the production of alcaligin siderophore or other iron receptor proteins are evidence of iron starvation in B. pertussis, and methods of evaluating the production levels of alcaligin siderophore or other iron receptor proteins in iron-starved B. pertussis cells are described herein and are known in the art. It would be appreciated that the cell culture may need to be processed in one or more ways prior to being combined with the pharmaceutically acceptable carrier. For example, the cells in the cell culture can be collected and washed to remove the iron-depleted media. Additionally or alternatively, iron-starved cells can be, for example, frozen or lyophilized.

In accordance with the present invention, there may be employed conventional molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims.

EXAMPLES Example 1 Production of Whole Cell Pertussis (wP) Vaccines

B. pertussis frozen stocks (in whole sheep blood, stored at −80° C.) were streaked onto Bordet Gengou agar plates and cultured at 37° C. for 48 h. Plate growth was suspended in complete Stainer-Scholte liquid medium, and used to inoculate seed cultures in complete Stainer-Scholte medium. After 36 to 48 h growth at 37° C. in a shaking incubator, bacteria were harvested by centrifugation, and the bacterial cells were washed at least twice using iron-free Stainer-Scholte medium. This washed seed suspension was used to inoculate complete Stainer Scholte medium (+Fe) or iron-free Stainer Scholte medium (−Fe) to an initial cell density corresponding to ˜2×10⁸ cfu/ml (˜0.1 OD₆₀₀), and grown for 36 to 48 h at 37° C. in a shaking incubator. Iron starvation status in iron-free cultures was confirmed by a siderophore detection assay. B. pertussis cells were harvested by centrifugation, and washed 3 times using cold sterile saline solution.

Bacteria were suspended in saline to an optical density representing ˜1×10⁹ cfu/ml (confirmed by plate counts), killed by heating at 65° C. for 0.5 h and adsorbed to alum (Alhydrogel®, 2%, InvivoGen) to yield +Fe and −Fe wP vaccine suspensions.

Example 2 Production of Recombinant B. pertussis Iron Receptor Proteins in E. coli

B. pertussis iron receptor genes were PCR-amplified from genomic DNA templates, and cloned into pBAD/His plasmid vectors (Invitrogen) for arabinose-inducible expression in E. coli. PCR primers were designed to include restriction site adapters for cloning, and to replace the receptors' start codons and N-terminal signal sequences with the plasmid vector-encoded start codon and polyhistidine tag for affinity purification of the products.

E. coli strain TOP10 (F-mcrA Δ(mrr-hsdRAIS-mcrBC) φ80lacZΔM15 ΔlacX74 mpG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(Str^(R)) endA1λ⁻) (Invitrogen) carrying recombinant pBAD/His plasmids was cultured overnight on Luria-Bertani agar plates supplemented with ampicillin (200 μg/ml), then sub-cultured into Luria-Bertani broth supplemented with ampicillin and grown at 37° C. in a shaking incubator until mid-exponential phase. Arabinose (20% w/v solution) was added to 0.002% final concentration to induce receptor gene expression, and the cultures were grown for an additional 4 h. Bacterial cells were recovered by centrifugation, washed using 1/10 culture volume STE (50 mM Tris pH 8, 100 mM NaCl, 1 mM EDTA), and resuspended in 1/10 culture volume STE before freezing the suspension overnight at −80° C.

Thawed cell suspensions were disrupted using a French pressure cell, and the insoluble cell fraction was recovered by ultracentrifugation. The insoluble material was washed using 1/10 culture volume of TE (10 mM Tris, 1 mM EDTA pH 8.0), followed by washing using 1/10 culture volume of water.

The insoluble material was then suspended in 1/10 culture volume of DB (100 mM NaH₂PO₄, 10 mM Tris, 8 M urea) at a pH 8.0, and any insoluble material was removed by centrifugation and discarded.

The supernatant fluid containing solubilized receptor proteins was transferred to a 50-ml conical tube containing Ni²⁺-charged Chelating Sepharose Fast-Flow beads (Amersham) equilibrated in DB (pH 8.0), and bound on a rotating platform for 1 h at room temperature. The beads were pelleted gently, and the supernatant fluid containing the unbound proteins was discarded. The beads were gently resuspended in DB (pH 6.3) to wash away loosely-bound proteins, then the beads were again pelleted gently and the supernatant fluid was discarded. After repeating the wash with DB (pH 6.3), the slurry was transferred to a disposable column fitted with a glass fiber support. The column bed was eluted 4 times, each time using ½ bed volume of DB (pH 5.9), and the eluates were collected separately. Elution was repeated, except using DB (pH 4.5), and, again, the eluates were collected separately. Eluate fractions were analyzed by SDS-PAGE for the presence of the receptor proteins. Peak fractions were pooled and the receptor proteins were precipitated by the addition of an equal volume of acetone at −20° C. After 30 min at −20° C., the protein precipitate was recovered by centrifugation, briefly air-dried, then dissolved in TE containing 8 M urea. After removing any insoluble material by centrifugation, the cleared protein solution was rapidly diluted into 10 volumes of 10 mM CHAPS in TE to renature the protein. The protein solution was cleared by centrifugation or using a centrifugal filter unit with a 0.22 μm filter. Soluble receptor protein concentration was determined using the Bradford assay.

Example 3 Construction of Recombinant Plasmids Encoding B. pertussis Fimbriae-Iron Receptor Protein Chimeras

The fim3 coding sequences with stop codon, and the fhaB promoter region were each PCR amplified using B. pertussis genomic DNA template and 5′-phosphorylated oligonucleotide primers. The resulting products were combined with SmaI-linearized plasmid vector pBBR1MCS-5 by the ligase cycling reaction using bridging oligonucleotides to yield recombinant plasmid pBB5/P (fhaB-fim3). This plasmid places fim3 expression under the control of the strong, well-characterized Bvg-dependent fhaB filamentous hemagglutinin promoter, thus circumventing the negative influence of the fim3 downstream repressive element on fim3 expression, and the polyC tract involved in fim3 phase variation. The plasmid construct was verified by nucleotide sequencing, and SDS-PAGE analysis confirmed that B. pertussis carrying plasmid pBB5/PfhaB-fim3 overproduces the Fim3 protein. Sub-regions of B. pertussis iron receptor genes encoding known or predicted extracellular loop domains of the receptor proteins were PCR-amplified using oligonucleotide primers with fim3 adapters. The products were recombined with fim3 by PCR megaprimer cloning atfim3 sites predicted to be permissive for foreign peptide insertion without interference with fimbrial assembly and maturation in B. pertussis. Plasmids encoding Fim3-iron receptor chimeric proteins were confirmed by nucleotide sequencing, then transferred to B. pertussis by mating.

TABLE 2 FauA extracellular loop domains Extra SEQ cellular Amino ID loop acids FauA amino acid residues NO  1 205-209 GSWDY  1  2 235-250 SQGDSYVHFLDTRRRT  2  3 274-312 NHSNGFGSGFPLFYSDGSRTDFNRSVANNAPWARQDTEA  3  4 336-375 TDGRYLMKHVYRGGYPDRHTGIIAAPPAFSNYDGNLDRDD  4  5 398-448 MSIDNHSDIQRYAMVGPAPAIGSFFDWRRAHIQEPSWADTLSPADDVRTKQ  5  6 472-492 SDWKTKQMYFGSRREYRIKNQ  6  7 518-537 QPQNARDTSGGILPPIKSKS  7  8 558-593 FQTRQDNLAQVIPGSSIPGFPNMQASRAASGAKVEG  8  9 615-633 FTTKDASGNPINTNHPRSL  9 10 658-679 QSRMYQAAASPRGNVEVEQDSY 10 11 701-711 NNLFDKKYYDQ 11

TABLE 3 BhuR extracellular loop domains Extra SEQ cellular Amino ID loop acids BhuR amino acid residues NO:  1 274-316 HDDTSWLLQAGTRNGHDLDNRADTGGYGSKRSQPSPEDYAQNN 12  2 338-367 FKRRADLDQMYQQGAGTSYQYGANRTHEET 13  3 397-442 QRLRLDSSQDARRTRDGRAYARPGDPYFYGYPSGPYGRSNSIQESI 14  4 468-515 YGNRTEQYSDGYDNCPAIPPGTPAPMGPRLCDMLHTNQADMPRVKGSQ 15  5 542-569 YEQKPQQGGGYQNNPNAGALPPSSSGGR 16  6 595-627 RAPSATELYTNYGGPGTYLRVGNPSLKPETSKG 17  7 648-660 NRYQNFIDKNVPL 18  8 680-690 TGLANRARVRI 19  9 714-730 AVGKDENTGQHLNSVPP 20 10 755-778 RRDDVQYPEASASARYADFQAPGY 21 11 806-836 DKKYWEAINVPTAGAIAIPRPLDWYNEPGRS 22

TABLE 4 PCR primers for production of DNA segments for ligase cycling assembly for construction of fim3-expressing plasmid pBB5/PfhaB-fim3 SEQ SEQ Product Target Forward ID Reverse ID size region primer NO primer NO: (bp) fhaB TACGTGCCGGACAGGGTTT 23 ATTCCGACCAGCGAAGTGAAG 24 195 promoter fim3 ATGTCCAAGTTTTCATACCCTGC 25 TCAGGGGTAGACGACGGAAA 26 615 CDS

TABLE 5 Bridging oligonucleotides for ligase cycling assembly with SmaI-cut plasmid vector pBBR1MCS-5 for construction of pBB5/PfhaB-fim3 SEQ ID Vector Sequence NO: pBBR1MCS-5 GCTCTAGAACTAGTGGATCCCCCTACGTGCCGGACAGGGTTT 27 vector/fhaB promoter fhaB promoter/ CTTCACTTCGCTGGTCGGAATATGTCCAAGTTTTCATACCCTGC 28 fim3 CDS fim3/ TTTCCGTCGTCTACCCCTGAGGGCTGCAGGAATTCGATATCA 29 pBBR1MCS-5 vector

TABLE 6 Fim3 regions predicted to be permissive for iron receptor loop domain insertion Predicted Fim3 Fim amino SEQ ID Amino acid Nucleotide permissive site acid residues NO: residues positions Site 1 PFDIKLKECPQALGAL 30  76-91 226-273 Site 2 SATKAKGVEFRLA 31 128-140 382-420

TABLE 7 PCR primers for amplification of BhuR extracellular loop domains 3 and 4, and insertion at Fim3 permissive sites 1 and 2 by the PCR megaprimer method Fim3 SEQ SEQ BhuR insertion ID ID loop site Forward primer NO Reverse primer NO 3 1 CCCTTCGACATCAAGCTGAAGCTGGAC 32 GGGCTCGAAATACAGCTTG 33 AGCTCGCAGGAC AGCGATTCCTGGATCGAGT TGCT 3 2 GGCAACCTGAGCACCGTGTCGCTGGAC 34 GATGTGCTGGCCGTTGAGG 35 AGCTCGCAGGAC TTCGATTCCTGGATCGAGT TGCT 4 1 CCCTTCGACATCAAGCTGAAGGAGCAG 36 GGGCTCGAAATACAGCTTG 37 TACTCGGACGGCTA AGCTGGCTGCCCTTGACCC G 4 2 GGCAACCTGAGCACCGTGTCGGAGCAG 38 GATGTGCTGGCCGTTGAGG 39 TACTCGGACGGCTA TTCTGGCTGCCCTTGACCC G

TABLE 8 B. pertussis Tohama I fhaB promoter region DNA sequence (SEQ ID NO: 40): TACGTGCCGGACAGGGTTTGATGGTTTGACTAAGAAATTTCCTACAAGT CTTGTATAAATATCCATTGATGGACGGGATCATTACTGACTGACGAAGT GCTGAGGTTTATCCAGACTATGGCACTGGATTTCAAAACCTAAAACGAG CAGGCCGATAACGGATTCTGCCGATTACTTCACTTCGCTGGTCGGAAT

TABLE 9 B. pertussis Tohama I fim3 DNA sequence (SEQ ID NO: 41): ATGTCCAAGTTTTCATACCCTGCCTTGCGCGCCGCGCTTATCCTTGCCGC CTCGCCCGTACTGCCAGCGCTGGCCAACGACGGCACCATCGTCATCACCG GCAGCATCTCCGACCAGACCTGCGTCATCGAAGAGCCCAGCACCCTCAAC CATATCAAGGTCGTGCAACTGCCCAAGATTTCCAAGAACGCGCTCAGGAA CGACGGCGACACCGCCGGCGCCACGCCCTTCGACATCAAGCTGAAGGAAT GCCCCCAGGCGCTGGGCGCGCTCAAGCTGTATTTCGAGCCCGGCATCACC ACCAACTACGACACGGGCGATCTGATTGCCTACAAGCAGACCTACAACGC ATCCGGCAACGGCAACCTGAGCACCGTGTCGTCCGCCACCAAGGCCAAGG GCGTGGAGTTCCGCCTGGCCAACCTCAACGGCCAGCACATCCGCATGGGC ACGGACAAAACCACGCAAGCCGCGCAAACCTTTACCGGCAAGGTCACCAA TGGCAGCAAGAGCTACACCCTGCGCTATCTCGCCTCGTACGTGAAGAAAC CCAAGGAAGATGTCGACGCGGCCCAGATCACCAGCTACGTCGGCTTTTCC GTCGTCTACCCCTGA

TABLE 10 B. pertussis Tohama I Fim3 amino acid sequence (SEQ ID NO:42): MSKFSYPALRAALILAASPVLPALANDGTIVITGSISDQTCVIEEPSTLN HIKVVQLPKISKNALRNDGDTAGATPFDIKLKECPQALGALKLYFEPGIT TNYDTGDLIAYKQTYNASGNGNLSTVSSATKAKGVEFRLANLNGQHIRMG TDKTTQAAQTFTGKVTNGSKSYTLRYLASYVKKPKEDVDAAQITSYVGFS VVYP

TABLE 11 Extracellular loop domains of three other Bordetella TonB-dependent receptor proteins Extra SEQ cellular Amino ID HemC loop acids Amino acid residues NO  1 100-122 DTEDVKIVLDGAPKGFEKYRQGS 43   2 156-231 DTKDAADLLPPGARFGALAKYGRHSNDGQDIYSVAL 44 YGRTRADGADGLLYANRRDGGDLRRPDGTRFAYSRNNQRS  3 253-290 SNAAGWQPFAAKRDDLPAPSQADIDRYGLTEAWRRKLV 45  4 320-418 ARSDTRQRDRRSSRASQSAFLGTLGNKSWVDYRDDR 46 FDLSNESHVALGTAEHVLLAGLRWHRHRRDTLMYYP PGRGEPDYNHGYFQPHYMPSGTQTVRS  5 441-458 VANTGRPNDAPRYNNPAP 47  6 481-485 KAARG 48  7 510-526 AKSNVSGSSRALRPERI 49  8 554-595 FRNRGKHEIFQRRGVACRGQAEGGAASDCPKPLSNYRNLPGY 50  9 624-643 RDASPRDPWGPRTWIAEIPP 51 10 668-693 VRRQDRSPTDGDPLAGYWALPKTAGY 52 11 717-738 DNLFNRPYHPYLGEAVSGTGRN 53 Extra cellular Amino BfeA loop acids Amino acid residues  1 191-203 YTNQPEDSREGNT 54  2 227-259 NKTNPDARDINAGHANTSDNGNPSTAGREGVIN 55  3 285-317 QGNLFAGDTMNNANSDFSDSLYGKETNAMYREN 56  4 340-369 TRNARQREGLAGGPEGAPTAGGYDTARLKN 57  5 395-430 LRESLEDPAGTRQTYTGGAIGGTAPADRDPKSRQTS 58  6 456-462 NSEFGSN 59  7 488-529 KAPNLYQSNPNYLLYSRGNGCLASQTNTNGCYLVGNEDLSPE 60  8 552-589 FRNDYRNKIVAGTDVQYRLANGARVLQWTNSGKAVVEG 61  9 615-628 KEKATGEPLSVIPE 62 10 652-675 YGKQEGPSTNVRTGVELNGDGRQT 63 11 705-728 DKQLYREGNASSAGAATYNEPGRA 64 Extra cellular Amino BP3077 loop acids Amino acid residues  1 ... ...  2 227-251 LKRRSSDYRVPDWPDGKLAGSYSES 65  3 274-339 LESKYGLPGHNHEYEGCHPHGSHLHCGGHDDHGHGH 66 DEHEEGEAEHDHGHEHGAGDVPYVKLRSNR  4 363-383 TDYRHDEIEGGQLGTRFQNRG 67  5 409-427 SDFRATGEEAFLPRSKTRA 68  6 452-467 QRVSPQSGAPASRTAG 69  7 493-524 RLPSAQELYADGVHLATNTYEIGDPGLDRETS 70  8 547-576 NRVKNYIYANTLDRYEDFRLIEYTQRDAEF 71  9 600-616 VRGRLTGGGGNLPRIPA 72 10 639-657 VYRQDDIAAYESSTPGYDM 73 11 680-704 NNLLNKLAFNHASFISTVAPLPGRS 74

Example 4 Purification of Fim3-Iron Receptor Chimeric Proteins Produced in B. pertussis

Fimbriae were extracted from B. pertussis cells recovered from Stainer-Scholte liquid cultures by incubation in a solution of 4 M urea in phosphate-buffered saline at 60° C. for 30 min with gentle agitation, followed by precipitation with 4% PEG 600. In addition, the fim3-iron receptor encoding insert DNA fragments of pBB5/PfhaB-fim3 derivatives were sub-cloned into plasmid vector pET-3a for inducible T7 promoter-directed expression and high-level production of chimeric proteins in E. coli.

Example 5 Construction of Recombinant Plasmids Encoding B. pertussis Iron Receptor-Flagellin Protein Chimeras

Polyvalent iron receptor antigens are produced using the FliC protein as a display scaffold. Individual B. pertussis receptor loop domain coding sequences are spliced in-frame into the dispensable region of fliC borne on a ColE1 plasmid (a pBR322 derivative). Construction of FliC-loop domain chimeras use PCR-generated loop-encoding DNA segments as megaprimers. Expression is driven from the tac promoter under LacI control, and the product is exported and assembled on the bacterial surface as flagella. Flagella (5-10% total cell protein) are easily purified after mechanical shearing from the bacterium and dissociation into monomers at reduced pH. Purified FliC monomers readily polymerize into filaments (ca. 20,000-30,000 monomers) in physiological pH solution by seeding with small flagellar fragments. Different FliC-receptor loop fusion protein monomers are produced in E. coli and purified from its flagella, then combined and assembled in vitro to produce polyvalent vaccine antigen polymers displaying multiple different iron receptor loop domains.

Example 6 Results

FIG. 1 are exemplary photographs showing that serum from culture-positive human donors (“pertussis patient serum”) reacts with Bordetella iron source receptors. Sera were from B. pertussis culture-positive children (aged 6 to 17 years old, previously vaccinated, n=12) and a control group of normal adult donors not known to have been infected with B. pertussis (n=10).

FIG. 2 shows results of mouse infection studies that evidence the importance of alcaligin siderophore and heme iron utilization systems to in vivo fitness of B. pertussis at different stages of infection. FIG. 2 shows that a mutant B. pertussis strain lacking the FauA and BhuR receptors was severely attenuated in the mouse pertussis model.

FIG. 4 are photographs showing the production of the polyhistidine-tagged iron receptor proteins in E. coli and purification by metal affinity chromatography. FauA is shown simply as an example. Note that the FauA-specific antiserum shows negligible cross-reactivity with other iron receptor proteins. Similar results were obtained for the BhuR heme receptor protein, demonstrating that the receptor proteins are immunogenic.

The plasmid construct shown in FIG. 5A directs expression of Fim3 from the strong Bvg-dependent fhaB promoter, thus eliminating the influence of the downstream repressive element in the Fim3 initial transcribed region and the Fim3 promoter poly(C)-tract effecting phase-variant expression. B. pertussis expresses the accessory genes necessary for fimbrial assembly and export.

In addition, two different BhuR receptor protein extracellular loop domains were each spliced into site 1 within the Fim3 fimbrial protein (FIG. 6) and the chimeric products were overproduced in E. coli. FIG. 7 shows that the FIG. 3-loop chimeric proteins (L3, L4) were strongly reactive with rabbit antisera raised to the purified recombinant BhuR receptor protein. This result indicates that immunization using a chimeric protein as described herein may elicit an anti-iron receptor immune response in a subject that can block colonization and/or enhance immune recognition and clearance of the B. pertussis.

It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

Disclosed are methods and compositions that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. 

What is claimed is:
 1. A chimeric polypeptide comprising at least one Bordetella antigenic polypeptide comprising an iron receptor protein or an antigenic portion thereof spliced into a Bordetella scaffold protein selected from a fimbrial protein, a flagellin protein, and combinations thereof.
 2. The chimeric polypeptide of claim 1, wherein the antigenic portion of an iron receptor protein comprises at least one extracellular domain.
 3. The chimeric polypeptide of claim 1, wherein the iron receptor protein is a TonB-dependent receptor protein or an antigenic portion thereof.
 4. The chimeric polypeptide of claim 3, wherein the TonB-dependent receptor protein is a ferric enterobactin siderophore (BfeA) receptor protein.
 5. The chimeric polypeptide of claim 1, wherein the iron receptor protein is a hemin or hemoprotein receptor or an antigenic portion thereof.
 6. The chimeric polypeptide of claim 5, wherein the hemin or hemoprotein receptor is a BhuR protein.
 7. The chimeric polypeptide of claim 1, wherein the iron receptor protein is a siderophore receptor or an antigenic portion thereof.
 8. The chimeric polypeptide of claim 7, wherein the siderophore receptor is an alcaligin siderophore receptor (FauA).
 9. The chimeric polypeptide of claim 1, wherein the fimbrial protein is a fimbrial 2 protein or fimbrial 3 protein.
 10. The chimeric polypeptide of claim 1, wherein the flagellin protein is a flagellin subunit protein.
 11. An acellular immunogenic composition, comprising the chimeric polypeptide of claim 1 and a pharmaceutically acceptable carrier.
 12. The acellular vaccine of claim 11, further comprising an adjuvant. 